The U.S. Government has rights in this invention pursuant to a grant from the National Institute of Health No. NIH-5-PO1-CA12174-12. This invention deals with the preparation of salts of acyl phosphoric acids, such as disodium acetyl phosphate. Such compounds are useful in the production of phosphorylating agents such as adenosine triphosphate (ATP), a required cofactor in many reactions in the biosynthesis of materials such as protein, carbohydrates, nucleotides, nucleic acids, and terpenes.
Biosynthesis by enzymatic catalysis has been attracting increased attention as a means of large scale production of many complex products. See, e.g., Skinner, "Enzymes Technology," Chem. & Engin. News, Vol. 53, No. 33, p. 22 (Aug. 18, 1975). A major limitation on the commercial usefulness of such processes, however, has been the cost of many of the cofactors or coenzymes necessary to conduct the reactions involved. For example, ATP is a cofactor which plays a prominent role in many biosynthetic processes, promoting formation of chemical bonds which otherwise would not form in significant quantities in dilute aqueous solutions. See generally, Stadtman, The Enzymes, Vol. 8, Chapter 1 3rd ed. 1972). For the use of ATP in cell-free enzymatic synthesis, see Baughn et al., J. Am. Chem. Soc., 100, 304 (1978), Wong et al., Methods Enzymol. 89, 108 (1982), all incorporated herein by reference. The high cost of cofactors such as ATP has severely inhibited the use of such processes on a commercial scale.
In order to reduce the cost of enzymatic synthesis requiring ATP, a system has been developed to regenerate ATP enzymatically from adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP). Normally, treatment of the raw materials used in biosynthesis with ATP to form more complex products results in the consumption of ATP and the production of AMP and/or ADP. Under the regeneration system, if AMP is produced in the biosynthesis, it is converted to ADP by enzyme catalyzed phosphoryl transfer from ATP according to the following equation: ##STR1## The ADP is then converted to ATP by reaction with a phosphate donor, e.g., acetyl phosphate (AcP), catalyzed by a phosphotransferase enzyme, e.g., acetate kinase: ##STR2## While this significantly reduced the difficulty in obtaining ATP, it did not eradicate it, largely because commercially acceptable methods of producing the phosphate donors such as acetyl phosphate were not known.
Acetyl phosphate had previously been synthesized from phosphoric acid by acylation with various ingredients, including acetyl chloride, ketene, isopropenyl acetate, and acetic anhydride, followed by isolation as the lithium or silver salts. See Whitesides et al., "Large-Scale Synthesis of Diammonium Acetyl Phosphate," J. Org. Chem., 40:2516 (1975) and refereces cited, incorporated herein by reference. All of these procedures involve difficult work-up and isolation sequences with low yields and/or expensive materials, and none of them are suitable for the preparation of acetyl phosphate in large quantity.
A simpler method for producing acyl phosphate salts, proposed in U.S. Pat. No. 4,088,675, includes acylation of phosphoric acid by a ketene, followed by precipitation of the acyl phosphate as an ammonium salt or a salt of certain organic bases. Further improvements in the process for synthesizing ammonium salts of acyl phosphate were suggested in the commonly assigned U.S. patent application Ser. No. 217,377 (Lewis et al., "Process for Producing Acyl Phosphate Salt," filed Dec. 6, 1980), now abandoned, but the resulting process still involved several steps which require careful experimental control, and which are accordingly difficult to carry out on a large scale. Other disadvantages include the requirement of anhydrous materials, such as 100% phosphoric acid which must be generated from the commercially available 85% phosphoric acid, and the requirement of filtration steps in the procedure. Further, the ammonium ion (used in this preparation to confer crystallinity to the solid product) has two disadvantages. First, it reacts with acetyl phosphate in solution. Second, it forms an insoluble precipitate (magnesium ammonium phosphate) under the reaction conditions. This precipitation both removes from solution the magnesium which may be required for activity of the enzymes [see, e.g., Mildvan, A.S. in The Enzymes, 2, 445, ed. Paul D Boyers (1970)], and occludes particles of immobilized enzyme. While the ammonium ion can be exchanged for another cation such as sodium by, for example, treatment with a cation exchange resin, this adds still another manipulation to the procedure, with a corresponding decrease in yield of the product.
There exists a need for a simple, convenient method for the production of acyl phosphate salt in a form that does not interfere with enzymatic reactions.